The overall goal of this project is to develop a series of facile, sensitive and reproducible high-throughput screening assays ready to identify potent, selective inhibitors of histone acetyltransferase (HAT) activity and apply these schemes to the initial development of potent and selective ySas2/hMOF HAT inhibitors. There are at least four families including Gcn5/PCAF, p300/CBP, MYST and Rtt109. HAT proteins use the acetyl-CoA cofactor to acetylate the 6 nitrogen of specific lysine residues within the N-terminal tails of core histones and work in concert with other histone modification enzymes to provide a mechanistic link between chromatin alteration and gene activation; and regulate genes involved in several different biological processes including cell cycle progression, dosage compensation and hormone signaling. Aberrant HAT function has also been correlated with several human diseases including various cancers (leukemia, melanoma, colorectal, gastric and lung), cardiovascular disease, obesity, HIV viral infection and drug addiction. Because of the interesting biology that is mediated by HATs and their relationship to human disease, it is of interest to develop novel small molecule inhibitors that might serve as biological probes as well as lead molecules for drug development. For this application, we will focus our screening efforts on the MYST HAT orthologs form yeast and human, ySas2 and hMOF, respectively, although the assay should be generally applicable to other HATs. The biological interest in ySas2/hMOF stems from it's evolutionarily conserved role in acetylating lysine 16 of histone H4 and the correlation of this activity with age-associated pathologies. The specific aims of the proposal are to (1) Develop a high-throughput screen for ySas2/hMOF inhibitors that can be automated to work in a 384-well plate format, (2) Define an orthogonal secondary assay to rapidly quantify the potency of candidate hit compounds identified in high-throughput screens, and (3) Develop counter-screening assays with representative HAT family proteins to assess compound selectivity. We expect these studies to define biochemical and cell based assays to screen large libraries of small molecules to identify potent and selective inhibitors of ySas2/hMOF HAT activity, and to be completely adaptable to screens for inhibitors of other HATs. We also anticipate that these studies will result in the identification of potent and selective first-generation ySas2/hMOF HAT inhibitors for use as chemical probes or further developed for therapeutic applications.